Many blood-sucking pests are known to attack humans and animals. Many of these are vectors for pathogenic microorganisms which threaten human health and commercially important livestock and pets. Various species of mosquitoes transmit diseases caused by viruses and many are vectors for disease-causing nematodes and protozoa. For example, mosquitoes of the genus Anopheles transmit malaria which causes approximately 1 million deaths annually. The mosquito species Aedes aegypti transmits an arbovirus that causes the disease yellow fever in humans. Other arboviruses transmitted by Aedes species include those that cause dengue fever, eastern and western encephalitis, Venezuelan equine encephalitis, St. Louis encephalitis, chikungunya, oroponehe and bunyarnidera. The genus Culex, which includes the common house mosquito C. pipiens, is implicated in the transmission of various forms of encephalitis and filarial worms. The common house mosquito also acts as a vector for Wuchereria banuffi and Brugia malayi, which are responsible for lymphatic filariasis. Trypanasomas cruzi, the causative agent of Chagas"" disease is transmitted by various species of blood-sucking Triatominae bugs. The tsetse fly (Glossina Spp.) acts as a vector for African trypanosomal diseases of humans and cattle. Many other diseases are transmitted by various blood-sucking pest species. Many of the blood-sucking pests are found within the order Diptera, including, for example, mosquitoes, black flies, no-see-ums (punkies), horse flies, deer flies and tsetse flies.
Various pesticides have been employed in efforts to control or eradicate populations of disease-bearing pests, such as disease-bearing blood-sucking pests. For example, DDT, a chlorinated hydrocarbon, has been used in attempts to eradicate malaria-bearing mosquitoes throughout the world. Other examples of chlorinated hydrocarbons, are BHC, lindane, chlorobenzilate, methoxychlor, and the cyclodienes (e.g., aldrin, dieldrin, chlordane, heptachlor, and endrin). The long-term stability and tendency of many of these pesticides to bioaccumulate render them particularly dangerous to the environment.
Another common class of pesticides is the organophosphates, which is perhaps the largest and most versatile class of pesticides. Organophosphates include, for example, parathion, Malathion, diazinon, naled, methyl parathion, and dichlorvos. Organophosphates are generally much more toxic than the chlorinated hydrocarbons. Their pesticidal effect is based on their ability to inhibit the enzyme cholinesterase, an essential enzyme in the functioning of the insect nervous system. However, they are also toxic to many animals, including humans.
The carbamates, a relatively new group of pesticides, include such compounds as carbamyl, methomyl, and carbofuran. These compounds are rapidly detoxified and eliminated from animal tissues. Their toxicity is thought to involve a mechanism similar to the mechanism of the organophosphates consequently they exhibit similar shortcomings, including animal toxicity.
A major problem in pest control results from the capability of many species to develop resistance. This resistance results from the selection of naturally occurring mutants possessing biochemical, physiological or behavioristic factors that confer some degree of immunity. Species of Anopheles mosquitoes have been known to develop resistance to DDT and dieldrin, the original pesticides used for house spraying. Substitute pesticides that are effective include Malathion, propoxur and fenitrothion; yet the cost of these pesticides is much greater than the cost of DDT.
Many pests, such as blood-sucking pests, require a proteinaceous meal to provide free amino acids that are necessary for egg development. The existence of oostatic hormones that inhibit digestion of the protein meal and thereby inhibit egg development has been demonstrated in various species, including house flies and mosquitoes.
In 1985, Borovsky purified an oostatic hormone 7,000-fold and disclosed that injection of a hormone preparation into the body cavity of blood imbibed mosquitoes caused inhibition of egg development and sterility (Borovsky, D. [1985] Arch. Insect Biochem. Physiol. 2:333-349). Following these observations, Borovsky (Borovsky, D. [1988] Arch. Ins. Biochem. Physiol. 7:187-210) disclosed that injection or passage of a peptide hormone preparation into mosquitoes inhibited the biosynthesis of serine esterase, trypsin-like and chymotrypsin-like enzymes in the epithelial cells of the gut, causing inefficient digestion of the blood meal and a reduction in the availability of free amino acids translocated by the hemolymph. Such amino acids are needed for the yolk protein synthesis in the fat body. When yolk protein is not synthesized yolk is not deposited in the ovaries, resulting in arrested egg development in the treated insect. It has been observed that the oostatic hormone peptides do not have an effect when inside the gut or other parts of the digestive system (Borovsky, D. [1988], supra).
In the mosquito Aedes aegypti, an early trypsin that is found in the midgut of newly emerged females is replaced, following the blood meal, by the late trypsin that is synthesized in a very short time; a female mosquito weighs 2 mg and produces 4 to 6 xcexcg trypsin within several hours after the blood meal. If trypsin would continue to be synthesized at this rate, female mosquitoes would spend all their energy on trypsin biosynthesis and would neither be able to mature their eggs nor find an oviposition site. To conserve energy the mosquito regulates trypsin biosynthesis with a hormone named Trypsin Modulating Oostatic Factor (TMOF). TMOF is synthesized in the follicular epithelium of the ovary 2-30 hours after a blood meal and is released in to the hemolymph, binding to a specific receptor on the midgut epithelial cells signaling the termination of trypsin biosynthesis. Mosquito larvae also synthesize trypsin as their major protease and use the enzyme to digest decaying organic material or small organisms like algae that are found in ponds and marshes.
Following the initial report by Borovsky in 1985, the isolated 10 amino acid hormone, trypsin modulating oostatic factor (TMOF) was isolated. TMOF (YDPAP6) (SEQ ID NO. 8) and two analogs (DYPAP6 and PAP6) (SEQ ID NOs. 9 and 10) of that peptide, were disclosed in U.S. Pat. Nos. 5,011,909 and 5,130,253, and in a 1990 publication (Borovsky, D., D. A. Carlson, P. R. Griffin, J. Shabanowitz, D. F. Hunt [1990] FASEB J. 4:3015-3020).
U.S. Pat. No. 5,358,934 discloses truncated forms of the full length TMOF which have prolines removed from the C terminus, including the peptides YDPAP (SEQ ID NO. 11), YDPAPP (SEQ ID NO. 12), YDPAPPP (SEQ ID NO. 13), and YDPAPPPP (SEQ ID NO. 14).
Neuropeptides Y (NPY) are an abundant family of peptides that are widely distributed in the central nervous system of vertebrates. In invertebrates members of NPY family have been recently isolated and their structures have been determined in a cestode and a turbellarian, respectively (Maule et al., 1991 xe2x80x9cNeuropeptide F: A Novel Parasitic Flatworm Regulatory Peptide from Moniezia expansa (Cestoda: Cyclophylidea)xe2x80x9d Parasitology 102:309-316; Curry et al., 1992 xe2x80x9cNeuropeptide F: Primary Structure from the Turbellarian, Arthioposthia triangulataxe2x80x9d Comp. Biochem. Physiol. 101C:269-274) and in terrestrial and marine molluscs (Leung et al., 1992 xe2x80x9cThe Primary Structure of Neuropeptide F (NPF) from the Garden Snail, Helix aspersaxe2x80x9d Regul. Pep. 41:71-81; Rajpara et al., 1992 xe2x80x9cIdentification and Molecular Cloning of Neuropeptide Y Homolog that Produces Prolonged Inhibition in aplysia Neuronsxe2x80x9d Neuron. 9:505-513). The invertebrate NPYs exhibit high homology to vertebrate NPYs at the carboxyl terminus. The major difference between vertebrate and invertebrate NPYs at the C-terminus is that the vertebrate NPY has an amidated tyrosine (Y) whereas invertebrates have an amidated phenyl alanine (F). Because of this difference, the invertebrate peptides have been named NPF.
Cytoimmunochemical analyses of the NPY family members suggest that they are concentrated in the brain of various insects (Verhaert et al., 1985 xe2x80x9cDistinct Localization of FMRFamide- and Bovine Pancreatic Polypeptide-Like Material in the Brain, Retrocerebal Complex and Subesophageal Ganglion of the Cockroach Periplaneta americanaxe2x80x9d L. Brain Res. 348:331-338) including the Colorado potato beetle Leptinotarsa decemlineata (Veenstra et al., 1985 xe2x80x9cImmunocytochemical Localization of Peptidergic Neurons and Neurosecretory Cells in the Neuro-Endocrine System of the Colorado Potato Beetle with Antisera to Vertebrate Regulatory Peptidesxe2x80x9d Histochemistry 82:9-18). Partial purification of the members of the NPY family in insects suggests that both NPY and NPF are synthesized in insects (Duve et al., 1981 xe2x80x9cIsolation and Partial Characterization of Pancreatic Polypeptide-like Material in the Brain of the Blowfly alliphora vomitoriaxe2x80x9d Biochem. J. 197, 767-770).
Recently two novel neuropeptides with NPF-like immunoreactivity have been isolated from brain extracts of the Colorado potato beetle. The peptides were purified using C18 reversed phase HPLC and their structure was determined using mass spectrometry. The deduced structures of these peptides are: Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-(ARGPQLRLRFamide) (SEQ ID NO. 1) and Ala-Pro-Ser-Leu-Arg-Leu-Arg-Phe-(APSLRLRFamide) (SEQ ID NO. 2) and were designated as NPF I and NPFII, respectively (Spittaels, Kurt, Peter Verhaert, Chris Shaw, Richard N. Johnston et al. [1996] Insect Biochem. Molec. Biol. 26(4):375-382).
The widespread use of pesticides has resulted in growing environmental and health care concerns about the use of pesticides. Many pesticides are detrimental to humans, either directly during application, or indirectly through residues in food, water and the environment. There is clearly a longstanding need in the art for pesticidal compounds which are specific and which reduce or eliminate direct and/or indirect threats to human health posed by currently available pesticides. There is, therefore, a need for environmentally compatible, biodegradable, pest-specific pesticides that can effectively deplete or eliminate pests.
The subject invention provides materials and methods for controlling pests. In a preferred embodiment the pests are agriculture pests, and, in particular, insects. Specifically exemplified herein are materials and methods for the control of insect larvae.
In a preferred embodiment, the subject invention concerns a plant cell transformed to express a polynucleotide encoding a pesticidal agent capable of inhibiting trypsin biosynthesis. Ingestion of the transgenic plant cell by a pest causes a decrease in trypsin synthesis in the gut of the pest. This decrease in trypsin synthesis drastically slows down the breakdown of food resulting in starvation, and eventually death of the pest. Pesticidal agents useful according to the subject invention include, but are not limited to, TMOF and functional equivalents thereof, NPF and functional equivalents thereof, and other agents identifiable by, for example, assays employing a TMOF receptor.
One embodiment of the present invention concerns a pesticide composition comprising a peptide having the formula:
A1A2A3A4A5Flxe2x80x83xe2x80x83(Formula I) (SEQ ID NO. 5)
wherein:
A1 is selected from the group consisting of Y, A, D, F, G, M, P, S and Y;
A2 is selected from the group consisting of A, D, E, F, G, N, P, S and Y;
A3 is selected from the group consisting of A, D, F, G, L, P, S and Y;
A4 is optionally present when A3 is present and is selected from the group consisting of A, F, G, L and Y;
A5 is optionally present when A4 is present and is selected from the group consisting of A, F, L and P;
Fl is a flanking region which is optionally present and is selected from the group consisting of: P, PP, PPP, PPPP (SEQ ID NO. 6), and PPPPP (SEQ ID NO. 7).
In a more specific aspect the peptide or protein comprises an amino acid sequence which consists essentially of the amino acid sequence of Formula I. In a preferred aspect, the peptide or protein lacks TMOF amino acids adjacent to the amino acid sequence of Formula I. In still another aspect, the peptide consists of the amino acid sequence of Formula I.
In various embodiments, either A3A4A5, A3A4A5Fl, A4A5, A4A5Fl, A5 or A5Fl are no present. Where A5 is not present, Fl may be attached directly to A4. Where A4A5 is not present, Fl may be attached directly to A3. Finally, where A3A4A5 is not present, Fl may be attached directly to A2.
Preferred peptides are selected from the group consisting of: AAP (SEQ ID NO. 16), ADP (SEQ ID NO. 17), ADPAP (SEQ ID NO. 18), APA (SEQ ID NO. 19), DAA (SEQ ID NO. 20), DF (SEQ ID NO. 21), DPA (SEQ ID NO. 22), DY (SEQ ID NO. 23), DYP (SEQ ID NO. 24), FAP (SEQ ID NO. 25), FDP (SEQ ID NO. 26), FDPAP (SEQ ID NO. 27), FSP (SEQ ID NO. 28), MPDYP5 (SEQ ID NO. 29), PAA (SEQ ID NO. 30), PAP (SEQ ID NO. 31), Y(D)DP (SEQ ID NO. 32), Y(D)DPAP (SEQ ID NO. 33), YAP (SEQ ID NO. 34), YD (SEQ ID NO. 35), YDA (SEQ ID NO. 36), YDAAP (SEQ ID NO. 37), YDF (SEQ ID NO. 38), YDFAP (SEQ ID NO. 39), YDG (SEQ ID NO. 40), YDLAP (SEQ ID NO. 41), YDP (SEQ ID NO. 42), (D)YDP (SEQ ID NO. 43), YDPAF (SEQ ID NO. 44), YDPAL (SEQ ID NO. 45), (D)YDPAP (SEQ ID NO. 46), YDPFP (SEQ ID NO. 47), YDPGP (SEQ ID NO. 48), YDPLP (SEQ ID NO. 49), YEPAP (SEQ ID NO. 50), YFPAP (SEQ ID NO. 51), YNPAP (SEQ ID NO. 52) and YSF (SEQ ID NO. 53).
A further embodiment of the present invention comprises a peptide having the formula
A1A2xe2x80x83xe2x80x83(Formula II) (SEQ ID NO. 63)
wherein
A1 is an amino acid selected from the group consisting of A, D, F, M, and Y, and
A2 is an amino acid selected from the group consisting of A, D, E, P, and Y.
In a preferred embodiment, the subject invention is directed to peptides of Formula II wherein A1 and A2 are independently selected from the group consisting of A, D, and Y.
Specifically exemplified as another embodiment are methods using an NPF peptide having the sequence Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-NH2 (SEQ ID NO. 1) or Ala-Pro-Ser-Leu-Arg-Leu-Arg-Phe-NH2 (SEQ ID NO. 2).
The biological control agents also comprise fragments, derivatives and analogs of NPF and TMOF peptides including, for example, NPF and/or TMOF peptides in which only conservative substitutions have been made. Analogs of the above-mentioned proteins and peptides which have one or more amino acid substitutions forming a branched peptide (e.g., by substitution with an amino acid or amino acid analog having a free amino- or carboxy-side chain that forms a peptide bond with a sequence of one or more amino acids, including but not limited to prolines) or allowing circularization of the peptide (e.g., substitution with a cysteine, or insertion of a cysteine at the amino- or carboxy-terminus or internally), to provide a sulfhydryl group for disulfide bond formation, are also provided.
The peptides of the present invention are particularly advantageous because their smaller size permits more rapid and efficient penetration into the midgut. In addition, they are less expensive to produce by conventional chemical methods.
In one embodiment, the subject invention provides pesticidal polypeptides having a C-terminus arginine. In a preferred embodiment, these short polypeptides can be joined to form polymers of repeating units. Specifically exemplified herein is the (DPAR)4 (SEQ ID NO. 61) polymer which can be broken into four DPAR (SEQ ID NO. 60) units in the gut of the pest. Advantageously, the short pesticidal polypeptides connected by arginine (or other readily cleavable residue) can penetrate the midgut of the pest efficiently.
Also included in this invention are addition salts, complexes, or prodrugs such as esters of the NPF and TMOF peptides, especially the nontoxic pharmaceutically or agriculturally acceptable acid addition salts. The acid addition salts can be prepared in standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic, ethanedisulfonic or methanesulfonic acids. Also, the N-terminus and C-termninus of the peptides can be chemically modified to further inhibit proteolysis by metabolic enzymes.
The NPF and TMOF peptides can also be synthesized wherein at least one of the amino acids is in the D-configuration, as opposed to the naturally occurring L-amino acids. The presence of D-configuration amino acids can inhibit the ability of proteases to degrade the peptides of the subject invention.
Also, derivation of these compounds with long chain hydrocarbons will facilitate passage through the cuticle into the pest body cavity. Therefore, a further embodiment of the subject invention pertains to compositions comprising the NPF and/or TMOF peptides bound to lipids or other carriers.
Yet another aspect of the subject invention pertains to polynucleotide sequences encoding the peptides disclosed herein. These DNA sequences can easily be synthesized by a person skilled in the art. The sequences may be used to transform an appropriate host to confer upon that host the ability to express the pesticidal peptides. Hosts of particular interest include bacteria, algae, yeasts, insect viruses, and plants. For each of these hosts, the polynucleotide sequences may be specifically designed by a person skilled in the art to utilize codons known to be optimally expressed in the particular hosts. Advantageous promoters can also easily be utilized. Bacteria, yeasts, plants, algae, viruses, and other hosts each may be used to produce peptides for further use, or these hosts can be used as vehicles for direct application of the peptide to the target pest. Plants can be transformed so as to make the plant toxic to a target pest species which feeds on that plant. Methods for transforming plant cells utilizing, for example agrobacteria, are well known to those skilled in the art.
The subject invention provides pest control compositions wherein the pest control agents are formulated for application to the target pests, or their situs. In a specific embodiment, recombinant hosts, which express a pest control agent are provided by the subject invention. The recombinant host may be, for example, procaryotic or eucaryotic.
Preferably, the subject peptides have an LD50 against pest larvae of less than 3.0 moles/ml. More preferably, the peptides have an LD50 of less than 2.0 moles/ml, and, most preferably, the peptides have an LD50 of less than 1.0 moles/ml. As used herein, xe2x80x9cLD50xe2x80x3xe2x80x9d refers to a lethal dose of a peptide able to cause 50% mortality of larvae maintained on a diet of 1 mg/ml autoclaved yeast (Borovsky and Mahmood [1995] xe2x80x9cFeeding the mosquito Aedes aegypti with TMOF and its analogs; effect on trypsin biosynthesis and egg development,xe2x80x9d Regulatory Peptides 57:273-281).
Another aspect of the subject invention relates to a plant comprising a plant cell transformed to express a polynucleotide encoding for a pesticidal agent of the subject invention. Further, the invention provides a plant tissue comprising a plant cell transformed to express a polynucleotide encoding a pesticidal agent of the subject invention.
A further aspect of the subject invention pertains to a method of increasing the pest-resistance of a plant comprising transforming a plant cell to express a polynucleotide encoding a pesticidal agent of the subject invention and culturing said plant cell. Preferably, the method further comprises regenerating a plant from the plant cell, wherein the plant comprises a plant cell expressing a polynucleotide encoding a pesticidal agent.
Yet an additional aspect of the subject invention pertains to a method of controlling agricultural pests comprising administering to the pests a pesticidal agent of the subject invention.
Still a further aspect of the subject invention concerns a method of controlling agricultural pests comprising transforming a microbe to express a polynucleotide encoding a pesticidal agent of the subject invention and administering the microbe to the pests.
The methods and materials of the subject invention provide a novel means for controlling agricultural pests and alleviating the destruction they can cause. In a preferred embodiment, the pesticidal agents of the subject invention disrupt the food digestion and egg production of the pests. Since the targets of the pesticidal agents can include receptors intrinsic to the survival of the pest, it will be very difficult for the pests to adapt and become resistant to the pesticidal materials and methods of the subject invention. This is a marked improvement over currently available agents and methods to which pests have already begun to develop resistance.
As used herein, the term xe2x80x9cpesticidally effectivexe2x80x9d is used to indicate an amount or concentration of a pesticide which is sufficient to reduce the number of pests in a geographical area, as compared to a corresponding geographical area in the absence of the amount or concentration of the pesticide.
The term xe2x80x9cpesticidalxe2x80x9d is not intended to refer only to the ability to kill pests, but also includes the ability to interfere with a pest""s life cycle in any way that results in an overall reduction in the pest population. For example, the term xe2x80x9cpesticidalxe2x80x9d includes inhibition or elimination of reproductive ability of a pest, as well as inhibition of a pest from progressing from one form to a more mature form, e.g., transition between various larval instars or transition from larva to pupa or pupa to adult. Further, the term xe2x80x9cpesticidalxe2x80x9d is intended to encompass all phases of a pest life cycle; thus, for example, the term includes larvicidal and ovicidal actions.
The word xe2x80x9ctransformxe2x80x9d is broadly used herein to refer to introduction of an exogynous polynucleotide sequence into a prokaryotic or eukaryotic cell by any means known in the art (including for example, direct transmission of a polynucleotide sequence from a cell or virus particle as well as transmission by infective virus particles) resulting in a permanent or temporary alteration of genotype and in an immortal or non-immortal cell.
The terms xe2x80x9cpeptide,xe2x80x9d xe2x80x9cpolypeptide,xe2x80x9d and xe2x80x9cproteinxe2x80x9d as used herein are intended to refer to amino acid sequences of any length.